The hair-cell stereocilia play a major role in hearing. The main protein component of hair-cell stereocilia is actin, organized in oriented filaments, some of them attached to the actin-rich cuticular plate. Changes in hair-cell actin filaments are common both in noise-induced and in drug-induced hearing loss. Despite the central importance of actin to the proper structure and function of hair cells, and the clear association of disruptions of actin-containing structures with hair-cell damage, little is known about the dynamics of actin turnover, or even of protein turnover in general, in hair cells. Using the new methodology of Multiple Imaging Mass Spectrometry during the tenure of a High Risk/High Impact grant, we have imaged and measured the percentage turnover of protein using a non radioactive marker, 15N leucine, in a variety of cochlear location: stereocilia, tectorial membrane, Reissner membrane spiral ganglion and temporal bone. The resolution of the mass images is easily 50 nm. Together with the high precision of the 15N/ 14N or 13C/12C isotopic ratios measurements, it provides the possibility to map the turnover of protein (actin) along stereocilia or other cochlear structures. The Multiple Imaging Mass Spectrometer (MIMS) that we used has been donated to Harvard and is installed in our laboratory. We propose to use the MIMS to studying the subcellular location and turnover rate of protein in the cochlea of chicks. Studies will be performed on chicks in normal conditions, after ototoxic insult, and during regeneration. Special attention will be given to the stereocilia and to the tectorial membrane. We will look for association between protein turnover and DNA replication, particularly during regeneration. The use of Multiple Imaging Mass Spectrometry may open up a whole new area of investigation into the cellular effects of drug- induced injury. Furthermore, the combination of heavy isotope labeling with Multiple Imaging Mass Spectrometry may find wide use in other areas of auditory and vestibular research.